1. Field of the Invention
The present invention relates to the determination of the presence or concentration of an analyte, such as a sugar, in a sample, using a labeled protein sensor.
2. Description of the Related Art
A bibliography follows at the end of the Detailed Description of the Invention. The listed references are all incorporated herein by reference.
Diabetes results in long-term health consequences including cardiovascular disease and blindness. These adverse long-term health consequences result from erratic levels of blood glucose in diabetics. To control the long-term complications associated with diabetes, blood glucose levels must be tightly regulated. This requires careful monitoring of blood glucose involving the unpleasant procedure of drawing blood.
The need for real-time measurement of blood glucose has resulted in efforts to develop non-invasive and minimally invasive methods to monitor blood glucose. A wide variety of methods have been proposed, including near infrared spectroscopy [1-3], optical rotation [4, 5], amperometric [6, 7], calorimetric [8, 9] and fluorescence detection [10-15]. In spite of intensive efforts, no method is presently available for non-invasive measurement of blood glucose.
Most glucose sensors that use biological elements for signal transduction use electrochemical or calorimetric detection of glucose oxidase activity. This method is associated with difficulties including the influence of oxygen levels, inhibitors in the blood, and problems with electrodes. In addition, detection results in consumption of the analyte which can cause difficulties when measuring low glucose concentrations. Electrochemical measurements are known to require frequent calibration, which is not acceptable for a continuous glucose monitor.
Using fluorescence, glucose can be measured using fluorophores which respond either to glucose or to proteins such as concanavalin A (ConA). Glucose assays based on proteins are typically competitive assays in which glucose disrupts the binding of ConA to a glucose containing polymer such as dextran. ConA binding to the glucose polymer is typically detected by resonance energy transfer (RET).
While a practical glucose sensor such as ConA-dextran may be used to successfully detect glucose by competitive RET assay, it is not completely reversible [13-15]. ConA and dextran form aggregates which with time become increasingly resistant to disruption by glucose. A sensor is not useful for glucose monitoring unless binding is reversible.
Another approach to developing a biosensor is to genetically engineer a protein for site-specific positioning of allosteric signal transducing molecules. Structural principles are used to take advantage of cooperative interactions between the signaling molecule and ligand binding. This technique has been applied to Maltose binding protein and Glucose/Galactose binding protein of Escherichia coli (GGBP) [16, 17]. Structural studies of GGBP reveal two domains, the relative positions of which change upon the binding of glucose [18]. Such conformational changes can be expected to result in spectral changes of environmentally sensitive probes, or changes in the transfer efficiency between donor and acceptor pairs covalently bound to the protein. Spectral changes of environmentally sensitive probes have been reported for GGBP [17].
However, there remains a need in the art for improved methods for determining the presence or concentration of glucose using fluorescent sensing molecules.